Description:

Advantages:

• ForthespecificdeterminationofIgGanti-B₂GP1antibodies
• Excellentclinicalcorrelation
• Colorcodedreagents
• Totalincubationtime:40minutes
• Convenient,costeffective
• Choiceofsingleormulti-pointcalibration

KitComposition:

Reagents

EachREAADSIgGAnti-β₂GPl96-MicrowellTestKitcontainsthefollowingreagents(volumesmayvarydependingonthekitsizeandconfiguration):

• 12x8stABIlizedβ₂GPl(fromhumanserum)coatedmicrowellswithframe
• 60mlSampleDiluentIV(blue-greensolution)
• 3vials(0.250ml)IgGβ₂GPlCalibratorSerum*(1-high,2-moderate,3-low)(human);seeviallabelforantibodyconcentrationinGunits.Calibrator2shouldbeusedwhenperformingsinglepointcalibration
• 0.250mlIgGβ₂GPlPositiveControlSerum*(human);seeviallabelforexpectedGunitrange
• 0.250mlNormalControlSerum*(human);seeviallabelforexpectedGunitrange
• 15mlanti-humanIgG(goat)HRP-ConjugatedAntibodySolution(bluesolution)
• 15mlOneComponentSubstrateSolution(TMBandH₂O₂);readytouse
• 15mlStoppingSolution(0.36Nsulfuricacid)
• 2bottles(30ml)WashConcentrate(33XPBS/Tween)

Storeat2–8°C.DoNotFreeze.

MaterialsRequiredbutnotSupplied

• ReagentgradewatertopreparePBSwashsolutionandtozeroorblanktheplatereaderduringthefinalassaystep
• Graduatedcylinders
• Precisionpipettorscapableofdeliveringbetween5μland1000μl,withappropriatetips
• Miscellaneousglasswareappropriateforhandlingsmallvolumes
• Flasksorbottles,1liter
• Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
• Disposablegloves
• Plate-reADIngspectrophotometercapableofreadingabsorbanceat450nm(650nmreferenceifdualbeam)
• Multichannelpipettorscapableofdeliveringto8wellssimultaneously
• Microdilutiontubesanda96-wellmicrodilutiontubeholderforsampledilutionsandrapiddeliverytomicrowellplate

MeasurementPrinciple:

AssayProcedure:

1. Theassaycanbeperformedwithasinglepointcalibration(Calibrator2)orafour-pointcalibrationcurve(Calibrators1,2,and3plussamplediluent/reagentblankasCalibrator4equalto0Gunits).Areagentblankcontrolshouldalsoberunwithboththesinglepointandmultipointcalibrationmethods.SampleDiluentwithoutserumorplasmaisaddedtothewell.Thiswellwillbetreatedthesameasacontrolorpatientsampleinsubsequentassaysteps.
2. Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebagprovided.
3. Preparea1:50dilutionofthecalibrators,controls,andpatientsamplesinsamplediluent(blue-greensolution);e.g.,10μlsampleaddedto490μlSampleDiluentequalsa1:50sampledilution.
4. Add100μlofdilutedcalibrators(includingthereagentblank/Calibrator4),controls,andpatientsamplestotheappropriatemicrowells.
5. Incubate15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptythesamplefluid.Donotallowsamplestocontaminateothermicrowells.
6. Wash4timeswithwashsolution.Eachwellshouldbefilledwithwashsolutionperwash.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnappingmotionofthewristtoshaketheliquidfromthewells.Toretainmicrowellmodulesduringwashing,theframemustbesqueezedatthetopandbottomofthelongersides.Blotonabsorbentpapertoremoveresidualwashfluid.Donotallowwellstodryoutbetweensteps.
7. Add100μlanti-humanIgGHRP-ConjugatedAntibodySolution(blue)tothewells.
8. Incubatefor15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptytheconjugatesolution.
9. Wash4timeswithwashsolution,asinstep6.Useasnappingmotiontodraintheliquidandblotonabsorbenttowelsafterthefinalwash.Donotallowthewellstodryout.
10. Add100μlOne-ComponentSubstratetoeachwellandincubatefor10minutesatroomtemperature.Addsubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwellswithpositivesamples.
11. 11.Add100μlStoppingSolution(0.36Nsulfuricacid)toeachwelltostoptheenzymereaction.BesuretoaddtheacidtothewellsinthesameorderandatthesamerateastheSubstratewasadded.BlueSubstratewillturnyellowandcolorlesssolutionwillremaincolorless.Blankorzerotheplatereaderagainstanairorawaterblankwell.ReadtheO.D.ofeachwellat450nm(and650nmreferenceifdualbeam).TheO.D.valuesshouldbemeasuredwithin5minutesoftheadditionoftheStoppingSolution.

ClinicalProcedure:

ClinicalSpecificity:serumsamplesfrommultiplehealthyblooddonorpopulationswereassayedforthepresenceofIgG,IgM,IgAanti-B2GPIantibodies.Specificitywasshowntobe100%,93%,and96%,respectivelyforthethreeisotypes.

ClinicalSensitivity:anunselectedSLEpopulationwastestedtodeterminetheclinicalsensitivityoftheanti-B2GPIassays.

Sensitivitywas23%forIgG,20%forIgMand25%forIgA.Theclinicalsensitivityoftheassayforthrombosiswasdeterminedbycomparinganti-B2GPItestresultsfromtwogroupsofselectedSLEpatients:Group1,withaclinicalhistoryofthrombosisand/orthrombocytopenia;andGroup2,withnohistoryofthrombosis(control).Theresultsareshownbelow:

Background:

Anti-phospholipidantibodiesareaheterogeneousgroupofimmunoglobulinsthatbindtoseveralanionicphospholipids,includingcardiolipinandphosphatidylserine.1,2Highserumlevelsofanti-phospholipidantibodiesarefrequentlydetectedinpatientswithautoimmune(e.g.,SLE)andnon-autoimmunediseases,aswellasinapparentlyhealthyindividuals.Theseantibodieshavebeenassociatedwithanincreasedriskforrecurrentarterialandvenousthromboticevents,thrombocytopenia,andfetalloss.Thesemanifestationsarethemainfeaturesoftheanti-phospholipidsyndrome(APS).

Mostautoimmuneanti-phospholipidantibodiesrequireaserumcofactor(β₂GPl)foroptimalbinding.Ithasbeenshownthatmanyanti-phospholipidantibodiesmayreacttoaneoepitopeformedontheβ₂GPlmoleculebytheinteractionbetweenthephospholipidandβ₂GPl.Mostassaysforantiphospholipidantibodiescontainbovineserumasthesourceofcofactor.Morerecently,ithasbeenshownthatthebindingofβ₂GPltothemicrowellsurfacemayproduceaneoepitopesimilartothatwhencombinedwithaphospholipidandtheresultswiththissystemshowedagoodcorrelationwiththeanti-phospholipidsyndrome.TheSEROlogicdetectionofanti-β₂GPlantibodiesprovidesenhancedclinicalsensitivityforthrombosis.TheREAADSAnti-β₂GPlELISATestKitusesthewellknownELISAformattodetectanti-β₂GPlantibodiesinhumanserum.

Patientswithpositivereactionstobothanti-phospholipidandanti-β₂GPlassaysweremorelikelytohaveclinicalcomplicationsthanthosepositiveforonlyone.HigherprevalenceandmeanserumlevelsofIgGanti-β₂GPlantibodieshavebeenreportedinautoimmunepatients.Inaddition,anti-β₂GPlantibodiesinSLEpatientscorrelatedwithclinicalmanifestationsofanti-phospholipidsyndrome.

Diapharma Group，Inc

Diapharma Group INC成立于1997年，研究人员具有多年临床以及研发经验，专注于血液病领域的试剂盒，试剂以及仪器，主要涵盖血栓形成、止血、血小板功能分析、细胞凋亡、生态毒理学等。

The Diapharma Group, Inc. formed on January 1, 1997 from Pharmacia Hepar, Inc. in Franklin, Ohio, and began as the exclusive US and Canadian distributor of the Chromogenix substrates and assays.

Over a quarter-century ago, Chromogenix developed the first chromogenic substrate technology under the former name, Kabi Diagnostica. Kabi later merged with Pharmacia. Some of Diapharma’s current staff worked inside the Chromogenix department within Pharmacia’s heparin manufacturing plant.

In 1998, Diapharma moved to West Chester, Ohio, where it remains today. Over the years, Diapharma expanded its product line to include a variety of hemostasis, cell death, platelet function, ecotoxicology, assays, reagents, antibodies and instruments from superior manufacturers.

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